Parasites of the genus Leishmania threaten the population of 98 countries on 5 continents [Alvar et al. 2012]. Leishmaniasis, a disease caused by parasitic protozoa Leishmania (including, for example. L. aethiopica, L. donovani, L. infantum, L. major, L. mexicana, L. tropica), occurs in intertropical zones of America and Africa and extends into the temperate zone of South America, Asia and southern Europe. Depending on body part that is affected by the disease, three types of leishmaniasis are distinguished: cutaneous, muco-cutaneous and visceral. It is assumed that 2 million people (1.5 million of cutaneous leishmaniasis, 0.5 million of visceral leishmaniasis) are newly infected per year and 20,000 to 40,000 people die from the disease per year, whereas recently the total number of affected people reaches 12 million worldwide [WHO, 2012]. Since the reporting of the disease is mandatory only in 32 of the 98 countries affected by leishmaniasis, a large part of the sick people is not recorded anywhere. The disease threatens not only the residents of endemic countries, but also travellers [Kobets et al. 2012] and military forces located in these areas. Effective vaccine against the infection does not exist and the drugs in use have many undesirable side effects. In addition, parasites became resistant against these drugs in many areas [Kobets et al. 2012].
The impact of leishmaniasis on the health of the population was strongly underestimated for many years. In the last 10 years, due to climatic and environmental changes, wars and other unknown effects the endemic areas have been heavily extended and the number of reported cases increased. In Europe, for example, leishmaniasis was previously restricted to the Mediterranean region, but now it has spread to northern Italy and southern Germany, where dozens of cases have been reported in people who did not travel outside said area [Kobets et al. 2012], and recently autochthonous cases of infected animals were reported from Hungary and Switzerland. What is alarming is the fact that Phlebotomus, sandfly that transmits Leishmania, spreads in a north-eastern direction faster than predicted by the existing models.
Not every person who becomes infected with Leishmania will develop a disease. In the Mediterranean region, for example, it is estimated that there is one clinical case for about 30-100 subclinical infections [Pampiglione et al. 1975]. This underreporting can have immense consequences for blood banks. The blood of donors living in endemic areas of Greece were seropositive for Leishmania in 15% of cases [Kyriakou et al. 2003], and in the case of donors from endemic regions of Spain (Balearic Islands) in 11% [Riera et al. 2008]. These asymptomatic infections may develop into severe clinical forms in patients with compromised immune system, e. g. AIDS patients. Co-infection with Leishmania parasites and HIV is becoming a serious health problem in many countries of the world, because HIV infection increases the risk of developing of visceral leishmaniasis by 100 to 2320 times, and vice versa, Leishmania infection increases the risk of onset of AIDS [Kobets et al. 2012].
Dogs infected with Leishmania represent a serious problem in veterinary medicine. Infected dogs usually exhibit significant symptoms. However, both diseased and asymptomatic dogs pose a risk to humans because they are a reservoir of parasites, which are transmitted to humans by insects. In some areas of Brazil up to 24% of dogs are infected with Leishmania [Coura-Vital et al. 2011], while in some parts of southern Europe, the presence of antibodies against the parasite shows that up to 34% of dogs encountered the infection [Kobets et al. 2012]. In recent years, leishmaniasis was observed in domestic dogs and cats in the USA [Petersen, 2009].
Safe and effective human vaccine against the disease does not exist. Similarly, there is no suitable and simple treatment without side effects [Kobets et al. 2012]. The drugs that are used to treat visceral leishmaniasis [Kobets et al. 2012] and which can be used also for treatment of cutaneous and muco-cutaneous leishmaniasis are: pentavalent antimonials, amphotericin B, liposomal amphotericin B, miltefosine and paromomycin. The oldest chemotherapeutics exploited for the treatment of visceral leishmaniasis are the antimony salts. At present, antimony derivatives sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime) are administered as intramuscular or intravenous injection. The disadvantage of these drugs is low clinical efficacy in some areas, the emergence of parasite resistance (up to 60% in the Indian state of Bihar), long duration of treatment (30 days), toxicity and high price. Other chemotherapeutic agents effective in the treatment of leishmaniasis are drugs that were originally developed for the treatment of other diseases. It is a medicine for fighting fungal infections, amphotericin B (AmBisome), a medicine for treating cancer, miltefosine (Impavido), and a broad-spectrum antibiotic, paromomycin. Even these drugs are not ideal. Only miltefosine may be administered per os, but its effectiveness is reduced, and in some cases the administration of miltefosine lead to a patient's death caused by side effects of a drug [Sundar et al. 2012]. Amphotericin B and liposomal amphotericin B require intravenous infusion and paromomycin is administered intramuscularly. Also these drugs have many side effects, they are, except paromomycin, expensive, and just one single mutation is enough to the parasites become resistant to miltefosine. The disadvantage is also the long duration of treatment [Kobets et al. 2012]. Disadvantages of single drugs can be partially reduced by combination therapy [van Griensven et al. 2010]. Successful treatment with a single dose of liposomal amphotericin B in India [Sundar et al. 2010] was described, but these promising results should be still confirmed [Edwards et al. 2011].
The parasitic protozoa of the genus Trypanosoma include T. brucei and T. cruzi, which cause sleeping sickness or Chagas' disease.
Sleeping sickness (also known as African trypanosomiasis) is a human disease caused by the protozoan Trypanosoma brucei and transmitted by a tse-tse fly (the flies). The disease is “endemic” in large areas of sub-Saharan Africa (including 36 countries and inhabited by about 60 million people). According to WHO, in 2009 in Africa about 30,000 new cases of the disease were reported. The disease exists in two forms, or is caused by two subspecies: Trypanosoma brucei gambiense in West and Central Africa and Trypanosoma brucei rhodesiense in eastern and southern Africa, the subspecies listed as the second is much more aggressive and faster-acting. For the treatment of sleeping sickness, depending on the stage of the disease, pentamidine, melarsoprol and suramin are used, however, these drugs exhibit a number of side effects, can cause anaemia or renal damage. Prevention of sleeping sickness is virtually impossible, because there is no effective vaccine.
Chagas disease (also known as American trypanosomiasis) is a tropical parasitic disease caused by the protozoan Trypanosoma cruzi, which is transmitted through biting by predatory blood-sucking triatomine bugs (also known as “kissing bugs”). Infected bug transmits the infection only in the case if it discharges infected faeces during the bite. However, the transmission may not occur only through bite of the kissing bugs. The disease can be transmitted by blood from one person to another (for example, during transfusion or organ transplantation, or during pregnancy infected mother can infect her unborn child). WHO estimates that 7-8 million people are infected with this parasite. The incubation period is one to four weeks (in case of infection by blood transfusion, the incubation period may be extended up to 6 weeks). The disease affects mainly the heart, intestine and brain. Medical treatment is effective only in the initial phase of disease and vaccination against disease does not exist. The drugs in use are Nifurtimox and Benznidazol, their disadvantage is considerable toxicity, both drugs cause serious adverse reactions of digestive and nervous system.
Trypanosoma brucei brucei causes disease in animals (e.g. horses, camels, water buffalo), but not in humans.
From all the above data it is apparent that there is a need for a new drug with activity against parasitic protozoa of Trypanosomatidae family, which would not require complicated treatment and had fewer side effects than currently used drugs, and would be available in the endemic areas.
Diphenyleneiodonium ([1,1′-biphenyl]-2,2′-diyliodonium, DPI) is a compound having the following formula:

It was shown in rats that diphenyleneiodonium sulphate prevented alcohol-induced liver damage [Kono et al. 2001]. WO 2007/080598 disclosed a pharmaceutical composition comprising DPI or salt thereof for inhibiting neointima proliferation and preventing restenosis. WO 2012/135588 disclosed the use of diphenyleneiodonium chloride as chemotherapeutic agents in the treatment of serous tumours.
The DPI was previously reported that it kills the malaria parasite Plasmodium falciparum (IC50=0.001-0.00006 μM) [Yuan et al. Nat Chem Biol 5: 765-771, 2009].
The activity of DPI against the parasites of Trypanosomatidae family was not known yet.